Diagnostic assays for proteins linked to the prognosis of cancer are important to develop to assist the clinicians in their assessment of appropriate modes of therapy. One of the proteins associated with cancer and whose mutation is often associated with the progression of the disease is the zinc finger DNA binding protein p53. In most cases, analysis of the levels of p53 has involved immunohistochemistry looking for cells in tumors with elevated levels of the protein. This assay does not measure the functional level of the protein, something generally correlated with the severity of the disease in a variety of cancers. Other functional assays for p53 have been developed, but in most cases are not amenable to the high throughput screening necessary for analysis of multiple tumor and tissue samples. In the past two years, we have developed a rapid and sensitive DNA binding assay for p53 that can be done in a microtiter plate format making it amenable to high throughput screening. For this proposal, we will modify that assay for screening human cell lines and analyze the changes in p53 DNA binding and interacting proteins in these cells. The specific project goals are: 1. Test p53 DNA binding assay with specific human cell extracts 2. Modify the p53 DNA binding assay to determine total p53 levels and distinguish native from mutant p53 3. Use SELDI-MS to determine proteins bound to p53 This work is the next stage in making this assay clinically relevant. The kinds of questions we intend to address with this proposal include how are the changes in p53 DNA binding correlated with different ratios of mutant and wild-type protein, does the specificity for a particular DNA sequence by p53 change in different cell lines and are there different proteins that interact with DNA bound and free p53. This information may provide alternative means for screening cell lines and tumors for diagnostic purposes as well as provide basic information about the interactions of this important tumor suppressor in human cells. These approaches are general enough to be applied to a number of DNA binding proteins associated with other important human diseases.